Earthworms and worm castings have been recommended for their
beneficial effects in increasing yields and suppressing soilborne diseases.
However, in a few cases, earthworm castings have been shown to harbor soilborne
pathogens. The research documents that earthworm castings used as an amendment
in soilless potting mixes at several organic farms in North Carolina were
contaminated with Phytophthora capsici and several Pythium
species. Phytophthora capsici and P. attrantheridium were
subsequently isolated from rotted roots of vegetable seedlings grown in the
potting mix. Commercial producers of earthworm castings should only use clean
plant material to maintain earthworms and earthworm castings should be
ascertained as pathogen-free before incorporation into plant growth media.

Introduction

Earthworm castings are an odorless, nutrient-rich, organic medium
that supports a diverse microbial community (2,3,5). Castings are also rich in
calcium humate, a binding agent that reduces desiccation of the castings and
promotes incubation and proliferation of beneficial organisms such as
Trichoderma spp., Pseudomonas spp., and mycorrhizal fungi
(6,8,10,11). Worm castings have been shown to suppress Verticillium wilt of
eggplant (7) and Fusarium wilt of cyclamen, iris, and tomato (8,10). Earthworms
disperse propagules of both beneficial and plant pathogenic soil microorganisms
(4), but less is known about the potential of earthworm castings to serve as a
source of soilborne pathogens in agricultural systems (5,13). Here we report the
presence of Phytophthora and Pythium species in earthworm castings
and their association with disease in soilless potting mixes.

In spring 2010, four growers in North Carolina experienced problems
in their organic vegetable production. The first grower noticed disease symptoms
in cucumber, tomato, and pepper transplants soon after seeding. Seedlings were
stunted with marginal leaf necrosis and some eventually died. Two weeks later,
vegetable transplants at three other grower facilities exhibited damping-off.
All growers had used the same lot of custom-blended soilless mix in their
transplant product. The mix was composed of 50% peat and 50% perlite, and did
not contain synthetic wetting agents or fertilizers; moreover, there were no
pathogens detected in these soilless mix. The mix had also been amended with
worm castings produced and purchased from same source.

A sample of the soil was initially sent to the Soil Testing
Laboratory of the North Carolina Department of Agriculture (NCDA), Raleigh, NC (www.ncagr.com/agronomi/sthome.htm), for chemical analysis. The report
indicated satisfactory nutrient levels. Therefore, soilborne pathogens were
investigated to determine if the earthworm castings were a source for pathogens
causing damping-off of the vegetables at these organic farms.

Isolation from Diseased Plant and Worm Casting

Traditional techniques such as media isolation and Rhododendron
baiting were employed. Molecular approaches involving DNA sequence analysis and
Blast search (1) were used for species identification of the pathogens. Root
lesions from five diseased plants were excised and surface-sterilized in a 0.5%
sodium hypochlorite solution for 5 min, placed on potato dextrose agar (PDA),
and incubated for 3 days at room temperature in the dark. Isolates were initially
obtained from all diseased plant roots. The representative isolates from
cucumber, tomato, and pepper were named Phytophthora735, 738, 739, and
Pythium735, 736, 739, respectively.

An indirect isolation method was used to recover oomycetes from the
worm castings. Rhododendron leaves were surface-sterilized in 0.5%
sodium hypochlorite for 3 min, air dried, and used as bait by cutting the leaves
at the mid-vein in a herringbone pattern. Three to four cut leaves were placed
into worm castings for 48 h, then set on moistened paper inside a container
(length 30 cm, width 26 cm, height 10 cm) and incubated at room temperature in
the dark for 3 days, to allow for potential growth of oomycetes. Each
rhododendron leaf was examined daily and pieces of leaf showing discoloration
were placed on PDA. Putative isolates of Phytophthora and Pythium,
based on morphological characteristics, were consistently recovered from
diseased roots and from the worm castings (Table 1).

The colony of Phytophthora capsici on PDA after 5
days culturing is white with ashen mycelium. P. capsici has long
pedicels, sporangia are spherical to elongate with a tapering base. Sporangia
have long pedicels ranging from 30 to 130 μm. Sporangia are papillate,
ellipsoid, and fusiform. The lengths and widths of sporangia range from 34 to 67
and 15 to 40 μm. P. capsici produces antheridia, oogonia, and oospores.
Antheridia range with the diameters from 11 to 20 μm. Oogonia are spherical with
diameters ranging from 22 to 52 μm. Oospores are plerotic, the wall thicknesses
of oospores ranges from 3 to 6 μm, and the diameters oospores ranges from 20 to
33 μm.

The colony of Pythium attrantheridium on PDA after 5
days culturing is cottony with hyaline, yellowish, and well-branched mycelium.
Pythium grew much faster than Phytophthora. The width of aerial
hyphae is from 3 to 5 μm. There are plentiful hyphal swellings. The width of
hyphal swellings is from 20 to 27 μm, Sporangia are globose with the length of
11 to 20 μm. Sporangia contain 6 to12 zoospores. Oogonia are terminal with the
diameter from 14 to 29 μm. Antheridia have a broad apical attachment with
oogonia. Oospores are plerotic or aplerotic with the diameter ranging from 14 to
20 μm, the thickness of oospore walls is 2 to 3 μm. Further species
identification was based on DNA sequence analysis.

Species Identification Using DNA Sequence Analysis

Species identification of the oomycetes was determined by comparing
of rRNA ITS fragment sequences. Mycelium (1-cm × 1-cm disks) from 3-day-old-cultures of Pythium and Phytophthora grown on PDA, which
were ground for 1 min with a drill in the presence of 50 µl DNA extraction
buffer (1M Tris pH 8, 5M NaCI, 0.5M EDTA, 10% SDS, sterile distilled water). The
extract was diluted 100 fold with distilled water and 2 µl was used for PCR
amplification. PCR conditions were 1 cycle of 94°C for 2 min; 30 cycles of 94°C
for 1 min, 65°C for 1 min, 72°C for 3 min; and a final extension at 72°C for 10
min.

A set of primers for amplification were developed from the rRNA
internal transcribed spacer (ITS) sequences. Various Phytophthora spp.
(120 representative sequences) and Pythium spp. (131 representative
sequences) were retrieved from GenBank (National Center for Biotechnology
Information, www.ncbi.nlm.nih.gov), aligned using ClaustalX (12).
Genus-specific primers were designed based on homologous regions specific to
Phytophthora and Pythium, respectively. These primers were designed to
target ITS1 sequences exhibiting the most variability in both genera. A 220 and
280 bp fragment was amplified in Phytophthora using primers (PhytoF:
CGCGGTATGGTTGGCTTCGGCTGAAC and PyhtoR: GCGGGTAATCTTGCCTGATATCAGGTCC) and
Pythium using primers (PythiF: CCTGCGGAAGGATCATTACCACAC, and PythiuR:
CGAGCCTAGACATCCACTGCTG), respectively. These shorter ITS1 fragments amplified by
the primers contained all the variability necessary to identify these isolates
to species. The genus-specific primers were used for amplification with six
different known Pythium and ten Phytophthora spp., the result
showed that all the identification based on DNA sequence analysis using Blast
search (1) agreed with the identification based on morphology. Isolates that
were baited from the worm castings were identified as Phytophthora capsici,
P. attrantheridium, P. spinosum, and P. intermedium
(Table 1). However, only P. capsici and P. attrantheridium were
recovered from diseased roots of the same plant for all vegetable transplants
including cucumber, tomato, and pepper. The representative isolates from one farm
were sequenced for species identification, after that, the rest of the isolates
were identified based on the morphology compared with the representative
isolates being sequenced.

Three isolates of P. capsici and three P.attrantheridium were chosen for pathogenicity test. One-month-old cucumber
(cultivar Corona), tomato (cultivar Rio Colorado), and bell pepper (cultivar
Camelot) seedlings were planted in the pots (four plants in each pot with two
replications) with soilless mix (50% peat and 50% perlite) amended with P.
capsici and P. attrantheridium at a rate of 1% (v/v contained
600 cfu/g of vermiculite). Innoculation was prepared following the
procedure described earlier (9). Noninoculated controls (2 pots with 8 plants)
were planted in the soilless mix without pathogens and subjected to the same
conditions. The inoculation test repeated twice. Inoculated plants and
un-inoculated plants were kept in a greenhouse with a temperature range from 22
to 25°C. After 15 days, diseases symptoms from leaves and roots were observed
with the similar damping-off symptoms on all inoculated plants but not on the
control plants. Microorganisms were re-isolated from two symptomatic plants,
which had the identical morphological features as the original isolate.

Summary

Based on colony morphology of isolates from diseased plants,
Phytophthora spp. and Pythium spp. were suspected to be the causal
agents of disease on vegetable transplants and identification which was
subsequently confirmed by Blast analysis (1) of DNA of representative isolates.
Furthermore, we found DNA sequences of isolates from diseased plants and worm
castings were identical (Table 1). Inoculation using isolates from diseased
plants or worm castings resulted in the same symptoms as observed at the
farms. The same isolates could be recovered from the inoculated plants, thereby,
completing Koch’s postulates.

Diseases on the vegetable transplants were caused by P. capsici
and P. attrantheridium. The results suggest that the
source of inoculum causing damping-off of transplants came from the worm
castings the growers used at their farms. Furthermore, these pathogens remain
viable and pathogenic after passage through earthworms as evidenced by the
pathogenicity trials. Therefore, care should be taken in amending soil with
earthworm castings. In this case, the worm castings producer used culled
vegetable plants as part of his feedstock for earthworm farming and these plant
tissues vegetables might have been infected with oomycete pathogens. We highly
recommend that commercial worm castings producers always use clean material for
earthworm farming in order to avoid contamination with soilborne plant
pathogens. Moreover, we propose that worm castings be inspected before
application since soilborne plant pathogens such as Phytophthora,
Pythium, and Fusarium spp. et al. are easily spread by earthworms and
worm castings.

Acknowledgements

This research was supported by the grant from the USDA Southern
Plant Diagnostic Network. We thank Ms. Jinping Sun for her technical assistance.
We would like to thank Ms. Stella Chang for editing the early version of the
manuscript. We also would like to specially thank Dr. Ned Tisserat for his
advice and editing the manuscript.